In vivo excision and amplification of large human genomic segments using Cre/loxP- and large T antigen/SV40 ori-mediated machinery

Abstract

In vivo excision and amplification of pre-determined, large genomic segments, directly from the genome of a natural host could be a novel cloning method with applications such as gap sequencing, gene therapy, and functional analysis of unknown genes in human cells. In this study, an in vivo excision and amplification system for human cells was devised with the Cre/loxP system of bacteriophage P1 and large T antigen/SV40 ori system of Simian virus 40. Trans-acting gene cre and large T antigen, each of which was under the control of a tetracycline responsive promoter, were inserted into the 5`- and 3`-UTR regions of the iNOS gene, respectively, by homologous recombination. Two loxP sequences, each of which serves as the recognition site for recombinase Cre, were also integrated unidirectionally into the 5`- and 3`-UTR regions of the iNOS gene. SV40 ori sequence, which serves as a conditional replication system, was inserted between the loxP sequences. The human cell line carrying the inserted elements was stably maintained until the excision and amplification functions are triggered by the induction of Cre and large T antigen, respectively. Upon induction by doxycycline, the 50-kb iNOS genomic region of human chromosome 17 flanked by two loxP sites was excised successfully.